Intraflagellar Transport Research Articles

CCDC38 is required for sperm flagellum biogenesis and male fertility in mice.

The sperm flagellum is essential for male fertility, and defects in flagellum biogenesis are associated with male infertility. Deficiency of coiled-coil domain-containing (CCDC) 42 (CCDC42) is specifically associated with malformation of mouse sperm flagella. Here, we find that the testis-specific protein CCDC38 interacts with CCDC42, localizing on the manchette and sperm tail during spermiogenesis. Inactivation of CCDC38 in male mice results in a distorted manchette, multiple morphological abnormalities of the flagella of spermatozoa and eventually male sterility. Furthermore, we find that CCDC38 interacts with intraflagellar transport protein 88 (IFT88), as well as outer dense fibrous 2 (ODF2), and the knockout of Ccdc38 reduces transport of ODF2 to the flagellum. Altogether, our results uncover the essential role of CCDC38 in sperm flagellum biogenesis, and suggest that some mutations of these genes might be associated with male infertility in humans.

Restriction of intraflagellar transport to some microtubule doublets: An opportunity for cilia diversification?

Cilia are unique eukaryotic organelles and exhibit remarkable conservation across evolution. Nevertheless, very different types of configurations are encountered, raising the question of their evolution. Cilia are constructed by intraflagellar transport (IFT), the movement of large protein complexes or trains that deliver cilia components to the distal tip for assembly. Recent data revealed that IFT trains are restricted to some but not all nine doublet microtubules in the protist Trypanosoma brucei. Here, we propose that restricted positioning of IFT trains could offer potent options for cilia to evolve towards more complex (addition of new structural elements like in spermatozoa) or simpler configuration (loss of some elements like in primary cilia), and therefore be a driver of cilia diversification. We present two hypotheses to explain how IFT trains could be restricted to some doublets, either by a triage process taking place at the basal body level or by the development of molecular differences between ciliary microtubules.

Ttc21b deficiency attenuates autosomal dominant polycystic kidney disease in a kidney tubular- and maturation-dependent manner

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multiprotein complexes. Deletion of several IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult autosomal dominant polycystic kidney disease (ADPKD) mouse models. However, deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, we investigate the role of global deletion of an IFT-A gene, Ttc21b, in juvenile and adult mouse models of ADPKD. Both juvenile (postnatal day 21) and adult (six months of age) ADPKD mice exhibited kidney cysts, increased kidney weight/body weight ratios, lengthened kidney cilia, inflammation, and increased levels of the nutrient sensor, O-linked β-N-acetylglucosamine (O-GlcNAc). Deletion of Ttc21b in juvenile ADPKD mice reduced cortical collecting duct cystogenesis and kidney weight/body weight ratios, increased proximal tubular and glomerular dilations, but did not reduce cilia length, inflammation, nor O-GlcNAc levels. In contrast, Ttc21b deletion in adult ADPKD mice markedly attenuated kidney cystogenesis and reduced cilia length, inflammation, and O-GlcNAc levels. Thus, unlike IFT-B, the effect of Ttc21b deletion in mouse models of ADPKD is development-specific. Unlike an IFT-A adaptor, deleting Ttc21b in juvenile ADPKD mice is partially ameliorative. Thus, our studies suggest that different microenvironmental factors, found in distinct nephron segments and in developing versus mature stages, modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a pathological feature of ADPKD.

Aberrant Multiciliogenesis in Idiopathic Pulmonary Fibrosis.

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B, and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of cilium genes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF.

Protocol for determining the average speed and frequency of kinesin and dynein driven Intraflagellar Transport (IFT) in C. elegans

Protocol for determining the average speed and frequency of kinesin and dynein driven Intraflagellar Transport (IFT) in C. elegans

Protocol for determining the average speed and frequency of kinesin and dynein driven Intraflagellar Transport (IFT) in C. elegans

Protocol for determining the average speed and frequency of kinesin and dynein driven Intraflagellar Transport (IFT) in C. elegans

Transient accumulation and bidirectional movement of KIF13B in primary cilia.

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein-2 motors. Nematodes additionally employ a cell-type-specific kinesin-3 motor, KLP-6, which moves within cilia independently of IFT and regulates ciliary content and function. Here, we provide evidence that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured immortalized human retinal pigment epithelial (hTERT-RPE1) cells. Anterograde and retrograde intraciliary velocities of KIF13B were similar to those of IFT (as assayed using IFT172-eGFP), but intraciliary movement of KIF13B required its own motor domain and appeared to be cell-type specific. Our work provides the first demonstration of motor-driven, intraciliary movement by a vertebrate kinesin other than kinesin-2 motors.

Genetic Interaction of Thm2 and Thm1 Shapes Postnatal Craniofacial Bone.

Ciliopathies are genetic syndromes that link skeletal dysplasias to the dysfunction of primary cilia. Primary cilia are sensory organelles synthesized by intraflagellar transport (IFT)—A and B complexes, which traffic protein cargo along a microtubular core. We have reported that the deletion of the IFT-A gene, Thm2, together with a null allele of its paralog, Thm1, causes a small skeleton with a small mandible or micrognathia in juvenile mice. Using micro-computed tomography, here we quantify the craniofacial defects of Thm2−/−; Thm1aln/+ triple allele mutant mice. At postnatal day 14, triple allele mutant mice exhibited micrognathia, midface hypoplasia, and a decreased facial angle due to shortened upper jaw length, premaxilla, and nasal bones, reflecting altered development of facial anterior-posterior elements. Mutant mice also showed increased palatal width, while other aspects of the facial transverse, as well as vertical dimensions, remained intact. As such, other ciliopathy-related craniofacial defects, such as cleft lip and/or palate, hypo-/hypertelorism, broad nasal bridge, craniosynostosis, and facial asymmetry, were not observed. Calvarial-derived osteoblasts of triple allele mutant mice showed reduced bone formation in vitro that was ameliorated by Hedgehog agonist, SAG. Together, these data indicate that Thm2 and Thm1 genetically interact to regulate bone formation and sculpting of the postnatal face. The triple allele mutant mice present a novel model to study craniofacial bone development.

SPECC1L deficiency causes shortened primary cilia and genetically interacts with IFT‐A deficiency to affect palatogenesis

Orofacial clefts are among the most common congenital malformations, affecting approximately 1 in 700 births. SPECC1L mutations in patients with syndromic cleft lip and/or palate cluster in the second coiled coil domain (CCD2), which facilitates interaction of SPECC1L with microtubules. We have shown that loss of SPECC1L in a null allele (Specc1l∆Ex4) resulted in perinatal lethality and delayed palatal shelf elevation, but no cleft palate (CP) or exencephaly at birth. However, mice with an in‐frame deletion of SPECC1L‐CCD2 exhibited CP and exencephaly, which are common manifestations of ciliopathies. Given that SPECC1L cytoplasmic expression includes localization around the cilia base and that SPECC1L regulates actin cytoskeleton, we investigated an association between SPECC1L and primary cilia. We observed that primary cilia were shortened relative to wild‐type in E13.5 Specc1l∆Ex4/∆Ex4 as well as Specc1l∆CCD2/∆CCD2 mutant palatal mesenchyme and epithelium. The cytoplasmic expression of SPECC1L around the cilia base was diminished in Specc1l∆CCD2/∆CCD2 cells. Thus, loss of SPECC1L association with microtubules appears sufficient to prevent its pericentriolar localization and to cause cilia shortening. Super‐resolution imaging for acetylated‐α‐tubulin and ciliary membrane protein, ARL13B, revealed bulbous structures in Specc1l mutant cilia, suggesting a defect in ciliary protein trafficking. Due to the overlapping phenotypes of shortened cilia, exencephaly and CP between the intraflagellar transport (IFT)‐A mouse mutant, Thm1aln/aln, and Specc1l∆CCD2/∆CCD2, we crossed these mutants to look for a genetic interaction. 18% of Specc1l∆CCD2/+, 0% of Thm1aln/+, and 33% of double heterozygous Specc1l∆CCD2/+;Thm1aln/+ embryos showed CP, confirming a genetic interaction. This was observed when the mother was Specc1l∆CCD2/+. However, remarkably, if the mother was Thm1aln/+, there was no CP incidence in Specc1l∆CCD2/+ and Specc1l∆CCD2/+;Thm1aln/+ embryos even though they had shortened cilia, suggesting Thm1aln/+ mothers provided a protective environment against CP phenotype. Consistent with our previous data, Specc1l∆Ex4;Thm1aln/+ mice resulted in no CP. These results suggest that shortened cilia inSpecc1l mutants do not cause but rather modulate CP phenotype. Overall, our studies indicate a novel role for SPECC1L in ciliogenesis, a novel genetic interaction between Specc1l andThm1, and a novel protective maternal genetic effect of Thm1 in palatogenesis.

The ciliary gene INPP5E confers dorsal telencephalic identity to human cortical organoids by negatively regulating Sonic hedgehog signaling.

Defects in primary cilia, cellular antennas that control multiple intracellular signaling pathways, underlie several neurodevelopmental disorders, but it remains unknown how cilia control essential steps in human brain formation. Here, we show that cilia are present on the apical surface of radial glial cells in human fetal forebrain. Interfering with cilia signaling in human organoids by mutating the INPP5E gene leads to the formation of ventral telencephalic cell types instead of cortical progenitors and neurons. INPP5E mutant organoids also show increased Sonic hedgehog (SHH) signaling, and cyclopamine treatment partially rescues this ventralization. In addition, ciliary expression of SMO, GLI2, GPR161, and several intraflagellar transport (IFT) proteins is increased. Overall, these findings establish the importance of primary cilia for dorsal and ventral patterning in human corticogenesis, indicate a tissue-specific role of INPP5E as a negative regulator of SHH signaling, and have implications for the emerging roles of cilia in the pathogenesis of neurodevelopmental disorders.

An integrative modeling approach reveals the 3D structure of the Intraflagellar Transport A (IFT-A) complex

An integrative modeling approach reveals the 3D structure of the Intraflagellar Transport A (IFT-A) complex

An integrative modeling approach reveals the 3D structure of the Intraflagellar Transport A (IFT-A) complex

An integrative modeling approach reveals the 3D structure of the Intraflagellar Transport A (IFT-A) complex

An integrative modeling approach reveals the 3D structure of the Intraflagellar Transport A (IFT-A) complex

An integrative modeling approach reveals the 3D structure of the Intraflagellar Transport A (IFT-A) complex

Long noncoding RNA lnc-H2AFV-1 promotes cell growth by regulating aberrant m6A RNA modification in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor in the oral and maxillofacial regions, and long noncoding RNAs (lncRNAs) play crucial roles in the occurrence and progression of HNSCC. The lncRNA lnc‐H2AFV‐1 was found to be upregulated in HNSCC tissues; however, the function of lnc‐H2AFV‐1 in regulating HNSCC proliferation and the potential molecular mechanism is unclear. The present study evaluated the expression of lnc‐H2AFV‐1 in HNSCC tissues using quantitative real‐time PCR (qPCR) and associated abundant lnc‐H2AFV‐1 expression with tumor size. Functionally, lnc‐H2AFV‐1 significantly promoted the proliferation of HNSCC cells in vitro and in vivo. Quantified N6‐methyladenosine (m6A) RNA methylation and dot blot assays revealed that total m6A methylation in HNSCC cells was accompanied by lnc‐H2AFV‐1 expression. Western blotting showed that the expression of methyltransferase‐like (METTL) 3 and METTL14 was consistent with that of lnc‐H2AFV‐1, whereas the expression of demethylase fat mass and obesity‐associated (FTO) was contrary to that of lnc‐H2AFV‐1. Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) and MeRIP‐qPCR revealed that lnc‐H2AFV‐1 overexpression led to the elevated expression and maximal m6A methylation of intraflagellar transport (IFT) 80 in HNSCC. In addition, METTL3/14 knockdown decreased IFT80 expression. Thus, our findings suggested that lnc‐H2AFV‐1 might be a biomarker that alters m6A modification by regulating the m6A methylases METTL3/14 and FTO and then mediating the downstream target IFT80 to promote HNSCC progression.

Global DNA Methylation Profiles in Peripheral Blood of WTC-Exposed Community Members with Breast Cancer.

Breast cancer represents the most common cancer diagnosis among World Trade Center (WTC)-exposed community members, residents, and cleanup workers enrolled in the WTC Environmental Health Center (WTC EHC). The primary aims of this study were (1) to compare blood DNA methylation profiles of WTC-exposed community members with breast cancer and WTC-unexposed pre-diagnostic breast cancer blood samples, and (2) to compare the DNA methylation differences among the WTC EHC breast cancer cases and WTC-exposed cancer-free controls. Gene pathway enrichment analyses were further conducted. There were significant differences in DNA methylation between WTC-exposed breast cancer cases and unexposed prediagnostic breast cancer cases. The top differentially methylated genes were Intraflagellar Transport 74 (IFT74), WD repeat-containing protein 90 (WDR90), and Oncomodulin (OCM), which are commonly upregulated in tumors. Probes associated with established tumor suppressor genes (ATM, BRCA1, PALB2, and TP53) were hypermethylated among WTC-exposed breast cancer cases compared to the unexposed group. When comparing WTC EHC breast cancer cases vs. cancer-free controls, there appeared to be global hypomethylation among WTC-exposed breast cancer cases compared to exposed controls. Functional pathway analysis revealed enrichment of several gene pathways in WTC-exposed breast cancer cases including endocytosis, proteoglycans in cancer, regulation of actin cytoskeleton, axon guidance, focal adhesion, calcium signaling, cGMP-PKG signaling, mTOR, Hippo, and oxytocin signaling. The results suggest potential epigenetic links between WTC exposure and breast cancer in local community members enrolled in the WTC EHC program.

Depletion of Ift88 in thymic epithelial cells affects thymic synapse and T-cell differentiation in aged mice.

Primary cilia are ubiquitous hair-like organelles, usually projecting from the cell surface. They are essential for the organogenesis and homeostasis of various physiological functions, and their dysfunction leads to a plethora of human diseases. However, there are few reports on the role of primary cilia in the immune system; therefore, we focused on their role in the thymus that nurtures immature lymphocytes to full-fledged T cells. We detected primary cilia on the thymic epithelial cell (TEC) expressing transforming growth factor β (TGF-β) receptor in the basal body, and established a line of an intraflagellar transport protein 88 (Ift88) knockout mice lacking primary cilia in TECs (Ift88-TEC null mutant) to clarify their precise role in thymic organogenesis and T-cell differentiation. The Ift88-TEC null mutant mice showed stunted cilia or lack of cilia in TECs. The intercellular contact between T cells and the "thymic synapse" of medullary TECs was slightly disorganized in Ift88-TEC null mutants. Notably, the CD4- and CD8-single positive thymocyte subsets increased significantly. The absence or disorganization of thymic cilia downregulated the TGF-β signaling cascade, increasing the number of single positive thymocytes. To our knowledge, this is the first study reporting the physiological role of primary cilia and Ift88 in regulating the differentiation of the thymus and T cells.

Proteomic Analysis of Endometrial Cancer Tissues from Patients with Type 2 Diabetes Mellitus.

Endometrial cancer (EC) is the most common form of gynecological cancer. Type 2 diabetes mellitus is associated with an increased risk of EC. Currently, no proteomic studies have investigated the role of diabetes in endometrial cancers from clinical samples. The present study aims to elucidate the molecular link between diabetes and EC using a proteomic approach. Endometrial tissue samples were obtained from age-matched patients (EC Diabetic and EC Non-Diabetic) during surgery. Untargeted proteomic analysis of the endometrial tissues was carried out using a two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF). A total of 53 proteins were identified, with a significant difference in abundance (analysis of variance (ANOVA) test, p ≤ 0.05; fold-change ≥ 1.5) between the two groups, among which 30 were upregulated and 23 downregulated in the EC Diabetic group compared to EC Non-Diabetic. The significantly upregulated proteins included peroxiredoxin-1, vinculin, endoplasmin, annexin A5, calreticulin, and serotransferrin. The significantly downregulated proteins were myosin regulatory light polypeptide 9, Retinol dehydrogenase 12, protein WWC3, intraflagellar transport protein 88 homolog, superoxide dismutase [Cu-Zn], and retinal dehydrogenase 1. The network pathway was related to connective tissue disorder, developmental disorder, and hereditary disorder, with the identified proteins centered around dysregulation of ERK1/2 and F Actin signaling pathways. Cancer-associated protein alterations such as upregulation of peroxiredoxin-1, annexin 5, and iNOS, and downregulation of RDH12, retinaldehyde dehydrogenase 1, SOD1, and MYL 9, were found in the EC tissues of the diabetic group. Differential expression of proteins linked to cancer metastasis, such as the upregulation of vinculin and endoplasmin and downregulation of WWC3 and IFT88, was seen in the patients with diabetes. Calreticulin and alpha-enolase, which might have a role in the interplay between diabetes and EC, need further investigation.

Primary Cilia and Their Role in Acquired Heart Disease.

Primary cilia are non-motile plasma membrane extrusions that display a variety of receptors and mechanosensors. Loss of function results in ciliopathies, which have been strongly linked with congenital heart disease, as well as abnormal development and function of most organ systems. Adults with congenital heart disease have high rates of acquired heart failure, and usually die from a cardiac cause. Here we explore primary cilia’s role in acquired heart disease. Intraflagellar Transport 88 knockout results in reduced primary cilia, and knockout from cardiac endothelium produces myxomatous degeneration similar to mitral valve prolapse seen in adult humans. Induced primary cilia inactivation by other mechanisms also produces excess myocardial hypertrophy and altered scar architecture after ischemic injury, as well as hypertension due to a lack of vascular endothelial nitric oxide synthase activation and the resultant left ventricular dysfunction. Finally, primary cilia have cell-to-cell transmission capacity which, when blocked, leads to progressive left ventricular hypertrophy and heart failure, though this mechanism has not been fully established. Further research is still needed to understand primary cilia’s role in adult cardiac pathology, especially heart failure.

Piezo1 and IFT88 synergistically regulate mandibular condylar chondrocyte differentiation under cyclic tensile strain

Objective(s)Mandibular condyle chondrocytes (MCCs) are exposed to various mechanical environments. Primary cilia, as a carrier for ion channels, can sense mechanical signals. Intraflagellar transport protein 88 (IFT88) is crucial for the assembly and function of primary cilia. Piezo1 is a mechanically activated ion channel that mediates mechanical signal transduction. This study aimed to identify the possible synergistic effect between Piezo1 and IFT88 in MCC differentiation during mechanical conduction. Materials and methodsConfocal immunofluorescence staining was used to reveal the Piezo1 localization. Small interfering RNA (siRNA) technology was used to knock down the expression levels of Piezo1 and IFT88. The chondrogenic differentiation ability of MCCs was evaluated by Alcian blue staining, and the early differentiation ability was evaluated by Western blot of SOX9 and COL2A1. ResultsConfocal immunofluorescence results showed that Piezo1 localized in the root of primary cilia. Without cyclic tensile strain (CTS) stimuli, Alcian blue staining showed that Piezo1 knockdown had a marginal effect on the chondrogenic differentiation of MCCs, while IFT88 knockdown inhibited the chondrogenic differentiation. The protein levels of SOX9 and COL2A1 decreased significantly with CTS stimuli. However, these protein levels were restored when Piezo1 was knocked down. In addition, IFT88 knockdown decreased the protein level of Piezo1 with or without CTS. ConclusionPiezo1 and IFT88 might play a synergistic role in regulating MCC differentiation under CTS stimuli.

MEIG1 determines the manchette localization of IFT20 and IFT88, two intraflagellar transport components in male germ cells

Sperm flagella formation is a complex process that requires cargo transport systems to deliver structural proteins for sperm flagella assembly. Two cargo transport systems, the intramanchette transport (IMT) and intraflagellar transport (IFT), have been shown to play critical roles in spermatogenesis and sperm flagella formation. IMT exists only in elongating spermatids, while IFT is responsible for delivering cargo proteins in the developing cilia/flagella. Our laboratory discovered that mouse meiosis expressed gene 1 (MEIG1), a gene essential for sperm flagella formation, is present in the manchette of elongating spermatids. IFT complex components, IFT20 and IFT88, are also present in the manchette of the elongating spermatids. Given that the three proteins have the same localization in elongating spermatids and are essential for normal spermatogenesis and sperm flagella formation, we hypothesize that they are in the same complex, which is supported by co-immunoprecipitation assay using mouse testis extracts. In the Meig1 knockout mice, neither IFT20 nor IFT88 was present in the manchette in the elongating spermatids even though their localizations were normal in spermatocytes and round spermatids. However, MEIG1 was still present in the manchette in elongating spermatids of the conditional Ift20 knockout mice. In the sucrose gradient assay, both IFT20 and IFT88 proteins drifted from higher density fractions to lighter ones in the Meig1 knockout mice. MEIG1 distribution was not changed in the conditional Ift20 knockout mice. Finally, testicular IFT20 and IFT88 protein and mRNA levels were significantly reduced in Meig1 knockout mice. Our data suggests that MEIG1 is a key protein in determining the manchette localization of certain IFT components, including IFT20 and IFT88, in male germ cells.